Single metallic nanoparticle imaging for protein detection in cells (original) (raw)
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Imaging single metal-nanoparticles in cells by photothermal interference contrast
We have developped a photothermal method for far-field optical detection of nanometer-sized metal particles, combining high-frequency modulation and polarization interference contrast. We can image gold colloids down to 5 nm in diameter, with a signal-to-noise ratio higher than 10. This is a considerable improvement over commonly used optical methods based on resonance plasmon scattering which, for background reasons, are limited to particles of more than about 40 nm in diameter. We also show that in addition to its intrinsic sensitivity, our photothermal method is totally insensitive to non-absorbing scatterers, as 10 nm nanoparticles can be imaged in cells.
Single-molecule imaging in live cell using gold nanoparticles
Methods in cell biology, 2015
Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with...
A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging
Nano Letters, 2013
Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Because of these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5 nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used green fluorescent proteins (GFPs) with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.
Optics Express, 2012
We introduce photothermal optical lock-in Optical Coherence Microscopy (poli-OCM), a volumetric imaging technique, which combines the depth sectioning of OCM with the high sensitivity of photothermal microscopy while maintaining the fast acquisition speed inherent to OCM. We report on the detection of single 40 nm gold particles with a 0.5 µm lateral and 2 µm axial resolution over a 50 µm depth of field and the three-dimensional localization of gold colloids within living cells. In combination with intrinsic sample contrast measured with dark-field OCM, poli-OCM offers a versatile platform for functional cell imaging.
Nano Letters, 2017
There is a need for biochemical contrast mediators with high signal-to-noise ratios enabling noninvasive biomedical sensing, for example, for neural sensing and protein−protein interactions, in addition to cancer diagnostics. The translational challenge is to develop a biocompatible approach ensuring high biochemical contrast while avoiding a raise of the background signal. We here present a concept where gold nanoparticles (AuNPs) can be utilized as a stimuli responsive contrast medium by chemically triggering their ability to exhibit multiphoton-induced luminescence (MIL) when performing multiphoton laser scanning microscopy (MPM). Proof-of-principle is demonstrated using peptidefunctionalized AuNPs sensitive to zinc ions (Zn 2+). Dispersed particles are invisible in the MPM until addition of millimolar concentrations of Zn 2+ upon which MIL is enabled through particle aggregation caused by specific peptide interactions and folding. The process can be reversed by removal of the Zn 2+ using a chelator, thereby resuspending the AuNPs. In addition, the concept was demonstrated by exposing the particles to matrix metalloproteinase-7 (MMP-7) causing peptide digestion resulting in AuNP aggregation, significantly elevating the MIL signal from the background. The approach is based on the principle that aggregation shifts the plasmon resonance, elevating the absorption cross section in the near-infrared wavelength region enabling onset of MIL. This Letter demonstrates how biochemical sensing can be obtained in far-field MPM and should be further exploited as a future tool for noninvasive optical biosensing.
International Journal of Nanomedicine, 2018
Background: We have introduced a novel method to quantify the intracellular refractive index (RI) of living cells and determine the molecular interaction of two interacting molecules using single particle spectroscopy. The advantages of this proposed technique over fluorescence-based imaging techniques is that it does not require any contrasting agent and it does not blink and bleach. Instead, our technique provides a non-destructive, non-invasive, high-resolution imaging of live cells. Methods: To verify our technique, we initially tested our approach for a dielectric medium where gold nanoparticles (AuNPs) were embedded in a polyvinyl alcohol (PVA) matrix, which was then extended to the cellular environment. In the dielectric medium, we identified the single particle and dimer and determined the interparticle distance of AuNPs using confocal laser scattering microscopy. We also determined the single particle RI from dark-field scattering microscopy images, which was confirmed with Mie theory and finite-difference time-domain (FDTD) simulated results. The single particle spectroscopy and microscopy technique was then extended to determine the intracellular RI and biomolecular interaction inside living cells using hyperspectral imaging and dark-field scattering microscopy. Results: The novelty of the paper lies in the demonstration of a direct and accurate method to probe the intracellular RI and molecular interaction focused on single particle analysis whereas previous demonstrations were based on AuNP ensembles. Optically acquired single particle and dimer images was verified by correlated SEM images also optical spectrum with analytical models and FDTD simulations for both the dielectric and cellular environment. We reported the interparticle distance of AuNPs inside HeLa cells and intracellular refractive index, which was also confirmed with Mie Theory and extensive FDTD simulations. Conclusion: Moreover, we believe that our in-depth plasmonic NP-based alternate imaging technique will provide a new insight in monitoring cellular dynamics and tracking the targeted NPs within live cells, enabling us to use plasmonic NPs as an intracellular biosensor.
Cell Imaging with Fluorescent Bi-Metallic Nanoparticles
JOURNAL OF ADVANCES IN CHEMISTRY
Last decades various imaging techniques have been applied in biological and biomedical research, such as magnetic resonance imaging, different types of tomography, fluorescence/bioluminescence, ultrasound, as well as multimodality approaches. Fluorescence imaging, especially in combination with nanoscale materials, is a very prospective tool for experiments in vivo and clinical applications due to its high temporal and spatial resolutions. Fluorescent nanoparticles (NPs), having ability to interact with biomolecules both on the surface of and inside the cells, may revolutionize the cell imaging approaches for diagnostics and therapy. In our investigation we report about new method of cell imaging with fluorescent bi-metallic NPs synthesized by chemical reduction of the relevant ions. As the model of living organism, the cells of yeast Hansenula polymorpha were used. All NPs in minimal concentration (up to 0.05 mM) was proved to be non-toxic for yeast cells. The NPs and NPs-modified ...
Small Gold Nanorods with Tunable Absorption for Photothermal Microscopy in Cells
Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2017
The synthesis, sorting, and characterization of monodisperse gold nanorods with dimensions around 10 nm in length and below 6 nm in diameter is reported. They display tunable plasmon resonance in the near infrared, a region where cellular absorption is reduced. A dual color photothermal microscope is developed to demonstrate that they are promising single molecule probes for bioimaging.
International Journal of Biomedical Imaging, 2007
We have synthesized and characterized gold nanoparticles (spheres and rods) with optical extinction bands within the "optical imaging window." The intense plasmon resonant driven absorption and scattering peaks of these nanoparticles make them suitable as contrast agents for optical imaging techniques. Further, we have conjugated these gold nanoparticles to a mouse monoclonal antibody specific to HER2 overexpressing SKBR3 breast carcinoma cells. The bioconjugation protocol uses noncovalent modes of binding based on a combination of electrostatic and hydrophobic interactions of the antibody and the gold surface. We discuss various aspects of the synthesis and bioconjugation protocols and the characterization results of the functionalized nanoparticles. Some proposed applications of these potential molecular probes in the field of biomedical imaging are also discussed.